Comparative analysis of right element mutant <it>lox </it>sites on recombination efficiency in embryonic stem cells

• Main Authors: Araki Masatake, Okada Yuka, Araki Kimi, Yamamura Ken-ichi
• Format: Article
• Language: English
• Published: BMC 2010-03-01
• Series: BMC Biotechnology
• Online Access: http://www.biomedcentral.com/1472-6750/10/29

<p>Abstract</p> <p>Background</p> <p>Cre-mediated site-specific integrative recombination in mouse embryonic stem (ES) cells is a useful tool for genome engineering, allowing precise and repeated site-specific integration. To promote the integrative reaction, a left element/right element (LE/RE) mutant strategy using a pair of <it>lox </it>sites with mutations in the LE or RE of the <it>lox </it>sequence has previously been developed. Recombination between LE and RE mutant <it>lox </it>produces a wild-type <it>lox</it>P site as well as an LE+RE double mutant <it>lox </it>site, which has mutations in both sides and less affinity to Cre, resulting in stable integration. We previously demonstrated successful integrative recombination using <it>lox</it>71 (an LE mutant) and <it>lox</it>66 (an RE mutant) in ES cells. Recently, other LE/RE mutant <it>lox </it>sites showing higher recombination efficiency in <it>Escherichia coli </it>have been reported. However, their recombination efficiency in mammalian cells remains to be analyzed.</p> <p>Results</p> <p>Using ES cells, we compared six RE mutant <it>lox </it>sites, focusing on their recombination efficiency with <it>lox</it>71. All of the RE mutant <it>lox </it>sites showed similar recombination efficiency. We then analyzed the stability of the recombined product, i.e., the LE+RE double mutant <it>lox </it>site, under continuous and strong Cre activity in ES cells. Two RE mutants, <it>lox</it>JTZ17 and <it>lox</it>KR3, produced more stable LE+RE double mutant <it>lox </it>than did the <it>lox</it>66/71 double mutant.</p> <p>Conclusion</p> <p>The two mutant RE <it>lox </it>sites, <it>lox</it>JTZ17 and <it>lox</it>KR3, are more suitable than <it>lox</it>66 for Cre-mediated integration or inversion in ES cells.</p>